Reuber H35 hepatoma cells are being used in studies of the mechanism by which cyclic AMP (cAMP) analogs regulate the synthesis of tyrosine aminotransferase (TAT) and PEP carboxykinase (PEPCK). Evidence to date has suggested that the cAMp analogs act as the level of translation. Ribosomal transit times for both protein will be determined by immunochemical precipitation of completed and polysome-bound nascent chains after brief labelling intervals in the presence and absence of cAMP analogs. Possible shifts in polysome size for TAT and PEPCK induced by cAMP analogs will also be examined by radioimmunochemical procedures. A series of 6- and 8-substituted cAMP analogs have been found to possess diverse abilities to induce tat and PEPCK. All these compounds, including some which are not inducers, are roughly equally capable of activating protein kinase in vitro, except N6,O2'-dibutyryl cAMP which is rather weak. In preliminary experiments with whole cells, protein kinase is activated (measured by f1 histone phosphorylation with 32Pi) so far only by analogs active as inducers. This analysis will be continued and kinetic considerations emphasized (i.e. does protein kinase activation precede increases in TAT/PEPCK synthesis?) cAMP-dependent ribosome-associated protein phosphorylation will also be examined and exploratory studies of the cell-free synthesis of both enzymes will be initiated. Somatic cell hybridization will be performed with H35 cells as one partner with a number of other cells, principally a human glioma cell time which is TAT minus but responds to PGE1 and catecholamines with a dramatic rise in cAMp concentration, which is not true of H35 cells. It is hoped that some of the hybrids generated will facilitate analysis of the mechanisms by which cAMP analogs specifically regulate the synthesis of these proteins.